scholarly journals Fnr Negatively Regulates Prodigiosin Synthesis in Serratia sp. ATCC 39006 During Aerobic Fermentation

2021 ◽  
Vol 12 ◽  
Author(s):  
Di Sun ◽  
Xuge Zhou ◽  
Cong Liu ◽  
Jingrong Zhu ◽  
Yunrui Ru ◽  
...  
Keyword(s):  
Oxygen Sensor ◽  
Anaerobic Metabolism ◽  
Model Organism ◽  
Transcriptional Regulator ◽  
Biosynthetic Gene Cluster ◽  
Biological Processes ◽  
Biosynthetic Gene ◽  
Global Regulator ◽  
Anaerobic Conditions ◽  
First Time

The well-known Crp/Fnr family regulator Fnr has long been recognized as an oxygen sensor to regulate multiple biological processes, including the switch between aerobic/anaerobic metabolism, nitrogen fixation, bioluminescence, infection, and virulence. In most cases, Fnr was found to be active under anaerobic conditions. However, its role in aerobic antibiotic metabolism has not yet been revealed. In this research, we report that in the model organism, Serratia sp. ATCC 39006, Fnr (Ser39006_013370) negatively regulates prodigiosin production by binding to the spacer between the −10 and −35 region in the promoter of prodigiosin biosynthetic gene cluster under aerobic conditions. Fnr was also shown to modulate the anti-bacterial activity and motility by regulating pathway-specific regulatory genes, indicating that Fnr acts as a global regulator in Serratia sp. ATCC 39006. For the first time, we describe that Fnr regulates antibiotic synthesis in the presence of oxygen, which expands the known physiological functions of Fnr and benefits the further investigation of this important transcriptional regulator.

scholarly journals Cyclofaulknamycin with the Rare Amino Acid D-capreomycidine Isolated from a Well-Characterized Streptomyces albus Strain

2021 ◽  
Vol 9 (8) ◽  
pp. 1609
Author(s):  
Liliya Horbal ◽  
Marc Stierhof ◽  
Anja Palusczak ◽  
Nikolas Eckert ◽  
Josef Zapp ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pyridoxal Phosphate ◽  
Cyclic Peptide ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Streptomyces Albus ◽  
Cluster Biosynthesis ◽  
First Time

Targeted genome mining is an efficient method of biosynthetic gene cluster prioritization within constantly growing genome databases. Using two capreomycidine biosynthesis genes, alpha-ketoglutarate-dependent arginine beta-hydroxylase and pyridoxal-phosphate-dependent aminotransferase, we identified two types of clusters: one type containing both genes involved in the biosynthesis of the abovementioned moiety, and other clusters including only arginine hydroxylase. Detailed analysis of one of the clusters, the flk cluster from Streptomyces albus, led to the identification of a cyclic peptide that contains a rare D-capreomycidine moiety for the first time. The absence of the pyridoxal-phosphate-dependent aminotransferase gene in the flk cluster is compensated by the XNR_1347 gene in the S. albus genome, whose product is responsible for biosynthesis of the abovementioned nonproteinogenic amino acid. Herein, we report the structure of cyclofaulknamycin and the characteristics of its biosynthetic gene cluster, biosynthesis and bioactivity profile.

scholarly journals Direct Rbs Engineering of the Biosynthetic Gene Cluster for Efficient Productivity of Violaceins in E. Coli

2020 ◽  
Author(s):  
Yuyang Zhang ◽  
Hongping Chen ◽  
Yao Zhang ◽  
Huifang Yin ◽  
Chenyan Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Gene Cluster ◽  
Large Scale ◽  
Value Added ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Fermentation Time ◽  
Engineering Strategy ◽  
First Time

Abstract Background: Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12472 to efficiently improve the fermentation titers.Results: Through four rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA-) reached 3269.7 μM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. Conclusion: To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

scholarly journals Identification of AflR Binding Sites in the Genome of Aspergillus flavus by ChIP-Seq

2020 ◽  
Vol 6 (2) ◽  
pp. 52
Author(s):  
Qing Kong ◽  
Perng-Kuang Chang ◽  
Chunjuan Li ◽  
Zhaorong Hu ◽  
Mei Zheng ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Binding Sites ◽  
Biosynthetic Gene Cluster ◽  
Binding Motif ◽  
Gene Ontology Analysis ◽  
Biosynthetic Gene ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Translational Start ◽  
First Time

We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp binding motif was a perfect palindromic sequence, 5′-CSSGGGWTCGAWCCCSSG’3′ with S representing G or C and W representing A or T. On closer examination, we hypothesized that the 18-bp motif sequence identified contained two identical parts (here called motif A and motif B). Motif A was in positions 8–18 on the upper strand, while motif B was in positions 11-1 on the bottom strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound to other genes outside the aflatoxin biosynthetic gene cluster.

scholarly journals Heterologous expression of the atypical tetracycline chelocardin reveals the full set of genes required for its biosynthesis

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Tadeja Lukežič ◽  
Špela Pikl ◽  
Nestor Zaburannyi ◽  
Maja Remškar ◽  
Hrvoje Petković ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Regulatory Protein ◽  
Model Organism ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Heterologous Host ◽  
Yield Increase ◽  
Tract Infections

Abstract Background Chelocardin (CHD) exhibits a broad-spectrum antibiotic activity and showed promising results in a small phase II clinical study conducted on patients with urinary tract infections. Importantly, CHD was shown to be active also against tetracycline-resistant Gram-negative pathogens, which is gaining even more importance in today’s antibiotic crisis. We have demonstrated that modifications of CHD through genetic engineering of its producer, the actinomycete Amycolatopsis sulphurea, are not only possible but yielded even more potent antibiotics than CHD itself, like 2-carboxamido-2-deacetyl-chelocardin (CD-CHD), which is currently in preclinical evaluation. A. sulphurea is difficult to genetically manipulate and therefore manipulation of the chd biosynthetic gene cluster in a genetically amenable heterologous host would be of high importance for further drug-discovery efforts. Results We report heterologous expression of the CHD biosynthetic gene cluster in the model organism Streptomyces albus del14 strain. Unexpectedly, we found that the originally defined CHD gene cluster fails to provide all genes required for CHD formation, including an additional cyclase and two regulatory genes. Overexpression of the putative pathway-specific streptomyces antibiotic regulatory protein chdB in A. sulphurea resulted in an increase of both, CHD and CD-CHD production. Applying a metabolic-engineering approach, it was also possible to generate the potent CHD analogue, CD-CHD in S. albus. Finally, an additional yield increase was achieved in S. albus del14 by in-trans overexpression of the chdR exporter gene, which provides resistance to CHD and CDCHD. Conclusions We identified previously unknown genes in the CHD cluster, which were shown to be essential for chelocardin biosynthesis by expression of the full biosynthetic gene cluster in S. albus as heterologous host. When comparing to oxytetracycline biosynthesis, we observed that the CHD gene cluster contains additional enzymes not found in gene clusters encoding the biosynthesis of typical tetracyclines (such as oxytetracycline). This finding probably explains the different chemistries and modes of action, which make CHD/CD-CHD valuable lead structures for clinical candidates. Even though the CHD genes are derived from a rare actinomycete A. sulphurea, the yield of CHD in the heterologous host was very good. The corrected nucleotide sequence of the CHD gene cluster now contains all gene products required for the production of CHD in a genetically amenable heterologous host, thus opening new possibilities towards production of novel and potent tetracycline analogues with a new mode of action.

scholarly journals Mutational analysis of a phenazine biosynthetic gene cluster in Streptomyces anulatus 9663

2012 ◽  
Vol 8 ◽  
pp. 501-513 ◽  
Author(s):  
Orwah Saleh ◽  
Katrin Flinspach ◽  
Lucia Westrich ◽  
Andreas Kulik ◽  
Bertolt Gust ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Mutational Analysis ◽  
High Resolution Mass Spectrometry ◽  
Streptomyces Coelicolor ◽  
Transcriptional Regulator ◽  
Biosynthetic Gene Cluster ◽  
Amide Bond ◽  
Biosynthetic Gene ◽  
New Type ◽  
Host Strains

The biosynthetic gene cluster for endophenazines, i.e., prenylated phenazines from Streptomyces anulatus 9663, was heterologously expressed in several engineered host strains derived from Streptomyces coelicolor M145. The highest production levels were obtained in strain M512. Mutations in the rpoB and rpsL genes of the host, which result in increased production of other secondary metabolites, had no beneficial effect on the production of phenazines. The heterologous expression strains produced, besides the known phenazine compounds, a new prenylated phenazine, termed endophenazine E. The structure of endophenazine E was determined by high-resolution mass spectrometry and by one- and two-dimensional NMR spectroscopy. It represented a conjugate of endophenazine A (9-dimethylallylphenazine-1-carboxylic acid) and L-glutamine (L-Gln), with the carboxyl group of endophenazine A forming an amide bond to the α-amino group of L-Gln. Gene inactivation experiments in the gene cluster proved that ppzM codes for a phenazine N-methyltransferase. The gene ppzV apparently represents a new type of TetR-family regulator, specifically controlling the prenylation in endophenazine biosynthesis. The gene ppzY codes for a LysR-type regulator and most likely controls the biosynthesis of the phenazine core. A further putative transcriptional regulator is located in the vicinity of the cluster, but was found not to be required for phenazine or endophenazine formation. This is the first investigation of the regulatory genes of phenazine biosynthesis in Streptomyces.

scholarly journals Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuyang Zhang ◽  
Hongping Chen ◽  
Yao Zhang ◽  
Huifang Yin ◽  
Chenyan Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Gene Cluster ◽  
Large Scale ◽  
Value Added ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Fermentation Time ◽  
Engineering Strategy ◽  
First Time

Abstract Background Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers. Results Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA−) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. Conclusions To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium

2017 ◽  
Author(s):  
Joana Martins ◽  
Niina Leikoski ◽  
Matti Wahlsten ◽  
Joana Azevedo ◽  
Jorge Antunes ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Cyclic Peptides ◽  
Biosynthetic Gene Cluster ◽  
The Novel ◽  
Tyrosine Residue ◽  
Biosynthetic Gene ◽  
Post Translational Modification ◽  
Biological Assays ◽  
Mass Spectrometric Data ◽  
Spectrometric Data

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. Anacyclamides are macrocyclic cyanobactins with a highly diverse sequence that are common in the genus <i>Anabaena</i>. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, anacyclamide D8P (<b>1</b>), from <i>Sphaerospermopsis</i> sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (<i>acy</i>) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in <i>Escherichia</i> <i>coli</i> established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of <b>1</b> using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound <b>1</b> was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium <i>Halomonas aquamarina</i> CECT 5000.<br>

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